Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures

Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of approximately 2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHO-DG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.     

Developmental control of nuclear size and shape by Kugelkern and Kurzkern

BACKGROUND: The shape of a nucleus depends on the nuclear lamina, which is tightly associated with the inner nuclear membrane and on the interaction with the cytoskeleton. However, the mechanism connecting the differentiation state of a cell to the shape changes of its nucleus are not well understood. We investigated this question in early Drosophila embryos, where the nuclear shape changes from spherical to ellipsoidal together with a 2.5-fold increase in nuclear length during cellularization. RESULTS: We identified two genes, kugelkern and kurzkern, required for nuclear elongation. In kugelkern- and kurzkern-depleted embryos, the nuclei reach only half the length of the wild-type nuclei at the end of cellularization. The reduced nuclear size affects chromocenter formation as marked by Heterochromatin protein 1 and expression of a specific set of genes, including early zygotic genes. kugelkern contains a putative coiled-coil domain in the N-terminal half of the protein, a nuclear localization signal (NLS), and a C-terminal CxxM-motif. The carboxyterminal CxxM motif is required for the targeting of Kugelkern to the inner nuclear membrane, where it colocalizes with lamins. Depending on the farnesylation motif, expression of kugelkern in Drosophila embryos or Xenopus cells induces overproliferation of nuclear membrane. CONCLUSIONS: Kugelkern is so far the first nuclear protein, except for lamins, that contains a farnesylation site. Our findings suggest that Kugelkern is a rate-determining factor for nuclear size increase. We propose that association of farnesylated Kugelkern with the inner nuclear membrane induces expansion of nuclear surface area, allowing nuclear growth.     

The Haplotype Map of the human genome: a revolution in the genetics of complex diseases

More than 99.9 % of the sequence is identical when comparing the DNA from two individuals. The remaining 0.1 % is responsible, along with other factors such as the environment, for the risk level of developing complex diseases (such as asthma, diabetes or cancer) or for the different pharmacological response to drugs. Despite the incredible advances in genomics in the past few years, identifying the variants involved remains difficult because of the prodigious amount of information to process. The recent completion of the Haplotype Map of the human genome has raised great hopes in the field as it is expected to help reduce the complexity of association studies and thus accelerate the discovery of genes associated with complex diseases. This review details the rationale behind the HapMap project, gives a summary of the results and also describes potential applications of the Haplotype Map.     

Effect of tumor necrosis factor alpha and infliximab on apoptosis of B lymphocytes infected or not with Epstein-Barr virus

Chronic inflammation and immunosuppressive therapies increase the risk of non-Hodgkin's lymphoma associated or not with Epstein-Barr virus (EBV) infection. A possible link between infliximab treatment and increased risk of lymphoma has been suggested. Indeed, infliximab induces apoptosis of monocytes and activated T lymphocytes, but its effect on B lymphocytes infected or not with EBV is unknown. Secreted tumor necrosis factor (TNF) alpha and the expression level of TNF receptor 1 (TNFR1) and TNFR2 were compared in EBV-positive and negative B-cell lines. The impact of TNFalpha and infliximab on apoptosis of EBV-positive cells was analyzed regarding the activity of NF-kappaB. Increased expression of TNFalpha in EBV-positive cells suggested that infliximab could affect their survival. However, TNFalpha or infliximab incubation had no effect on apoptosis of EBV-positive cells. Loss of NF-kappaB activity sensitized lymphoblastoid cell lines to TNFalpha-induced apoptosis, but no direct effect of infliximab on apoptosis was detected. On the basis of our in vitro data, neither TNFalpha nor infliximab has a direct effect on apoptosis of B lymphocytes and EBV-positive cell lines. Thus, if an increased incidence of lymphoma were induced by TNFalpha blockers, it would not involve a direct effect on B cells but rather an impaired immune surveillance by T cells.     

New amphiphilic lactic acid oligomer-hyaluronan conjugates: synthesis and physicochemical characterization

The "grafting onto" strategy was used to conjugate DL-lactic acid oligomers (OLA) to hyaluronan (HA) for the sake of developing novel degradable HA-based self-assembling polymeric systems. Grafting was achieved by reacting COCl-terminated OLA with cetyltrimethylammonium hyaluronate (CTA-HA) in dimethyl sulfoxide (DMSO). The resulting CTA-HAOLA conjugates were purified and turned to sodium form (Na-HAOLA) by dissolution in a phosphate buffer-DMSO mixture and successive dialyses against DMSO, ethanol, and water. In contrast, when the same protocol was applied to CTA-HAOLA, phase separation with gel formation was observed. The solution phase was composed of Na-HAOLA whereas the gel phase was made of mixed CTA-Na-HAOLA salt with ca. 25% of the carboxyl groups neutralized by CTA. Gelation was assigned to intramolecular hydrophobic associations between OLA and cetyl alkyl chains that complemented electrostatic interactions between CTA and HA COO- groups synergistically. Therefore, the corresponding stabilized CTA ions required more drastic conditions to be released. Under the selected dialysis conditions, the CTA-Na-HAOLA gels formed tiny tubes. Na-HAOLA and CTA-Na-HAOLA were characterized by FTIR, one-dimensional 1H and two-dimensional 1H NMR. The extent of grafting was ca. 5% per disaccharidic repeating unit, regardless of the molecular weight, as determined by NMR and capillary zone electrophoresis. Amphiphilic Na-HAOLA molecules were aggregated and formed spherical species in water according to size exclusion chromatography combined with multiangle laser light scattering detection. The critical aggregation concentration ranged between 0.2 and 0.35% (w/v), depending of the molecular weight of the parent hyaluronan.     

The Advantages of Demographic Change after the Wave: Fewer and Older,but Healthier,Greener, and More Productive?

Population aging is an inevitable global demographic process. Most of the literature on the consequences of demographic change focuses on the economic and societal challenges that we will face as people live longer and have fewer children. In this paper, we (a) briefly describe key trends and projections of the magnitude and speed of population aging; (b) discuss the economic, social, and environmental consequences of population aging; and (c) investigate some of the opportunities that aging societies create. We use Germany as a case study. However, the general insights that we obtain can be generalized to other developed countries. We argue that there may be positive unintended side effects of population aging that can be leveraged to address pressing environmental problems and issues of gender inequality and intergenerational ties.